PaCS Is a Novel Cytoplasmic Structure Containing Functional Proteasome and Inducible by Cytokines/Trophic Factors

Abstract

A variety of ubiquitinated protein-containing cytoplasmic structures has been reported, from aggresomes to aggresome-like induced structures/sequestosomes or particle-rich cytoplasmic structures (PaCSs) that we recently observed in some human diseases. Nevertheless, the morphological and cytochemical patterns of the different structures remain largely unknown thus jeopardizing their univocal identification. Here, we show that PaCSs resulted from proteasome and polyubiquitinated protein accumulation into well-demarcated, membrane-free, cytoskeleton-poor areas enriched in glycogen and glycosaminoglycans. A major requirement for PaCS detection by either electron or confocal microscopy was the addition of osmium to aldehyde fixatives. However, by analyzing living cells, we found that proteasome chymotrypsin-like activity concentrated in well-defined cytoplasmic structures identified as PaCSs by ultrastructural morphology and immunocytochemistry of the same cells. PaCSs differed ultrastructurally and cytochemically from sequestosomes which may coexist with PaCSs. In human dendritic or natural killer cells, PaCSs were induced in vitro by cytokines/trophic factors during differentiation/activation from blood progenitors. Our results provide evidence that PaCS is indeed a novel distinctive cytoplasmic structure which may play a critical role in the ubiquitin–proteasome system response to immune, infectious or proneoplastic stimuli.

Chaperone molecules concentrate together with the ubiquitin–proteasome system inside particulate cytoplasmic structures: possible role in metabolism of misfolded proteins

“…Resin blocks from human gastric mucosal biopsies, human neoplasms from ovary, kidney, lung, stomach, intestine, pancreas, thyroid, brain and hematopoietic tissue neoplasms, and fetal human (age 11-20 weeks) tissue, or from cultured cell lines such as HeLa, SH-SY5Y neuroblastoma, and HL-60 promyelocytic leukemia were all available from previous studies (Necchi et al 2010(Necchi et al , 2011Sommi et al 2013). All specimens had been fixed for 4 h at 4 °C with 2 % formaldehyde/2.5 % glutaraldehyde in 0.2 M cacodylate or 0.1 M phosphate buffer (pH 7.3), followed by fixation in 1.5 % osmium tetroxide for 1 h at room temperature and embedding in Epon-Araldite.…”

“…Tests to evaluate the specificity of immunogold labeling were routinely carried out by omitting the specific antibodies in the first layer of the procedure or by substituting for them nonimmune IgG or IgM (Santa Cruz Biotechnology) or unrelated antibodies. Many antibodies had already been characterized in combined immunofluorescence and ultrastructural investigations of aldehyde-fixed tissues with or without osmication (Necchi et al 2010(Necchi et al , 2011Sommi et al 2013). In addition, the antibodies used in the present study were tested by immunoblotting of HeLa and SH-SY5Y cell lysates.…”

“…We recently described a novel cytoplasmic structure characterized by accumulation of barrel-like particles resembling ultrastructurally those of the proteasome complex and by selective immunoreactivity for proteasomal and ubiquitinated proteins (Necchi et al 2010;Sommi et al 2013). This particulate cytoplasmic structure (PaCS) was observed in a variety of epithelial and nonepithelial cell cultures and in cells and tissues ex vivo, particularly under neoplastic, preneoplastic, or inflammatory conditions (Necchi et al 2011(Necchi et al , 2012 and during development and differentiation of fetal cells .…”

“…PaCS showed ultrastructural and/or cytochemical differences from other ubiquitin-reactive cytoplasmic structures (Solcia et al 2014) such as: aggresomes (Garcia-Mata et al 2002;Johnston et al 1998,); juxtanuclear quality control compartment (JUNQ; Kaganovich et al 2008); dendritic cell aggresome-like induced structure (DALIS; Lelouard et al 2002;Herter et al 2005;Kondylis et al 2013), also seen in macrophages (Canadien et al 2005;Solcia et al 2014); ALIS of cells other than professional antigen-presenting cells (Szeto et al 2006); sequestosomes (Bjørkøy et al 2005;Sommi et al 2013); insoluble protein deposit (Kaganovich et al 2008); or the inclusion bodies found in pathological conditions such as neurodegenerative, muscular, and hepatocellular diseases (Kuusisto et al 2001;Zatloukal et al 2002). It has been proposed that PaCSs are involved in metabolism of misfolded proteins through their ubiquitin-proteasome system (UPS) activity (Necchi et al Abstract Ubiquitin-proteasome system (UPS) proteins and proteolytic activity are localized in a recently identified cytoplasmic structure characterized by accumulation of barrel-like particles, which is known as the particulate cytoplasmic structure (PaCS).…”

“…Present findings point to PaCSs as an integrated, active UPS center well equipped for metabolism of misfolded proteins, especially in cells under physiological (fetal development) or pathological (neoplasia or inflammation) stress. 2011Sommi et al 2013). However, the accumulation of polyubiquitinated proteins in PaCSs also suggests some malfunction or insufficiency of the 26S proteasome component, which is required for rapid degradation of such proteins (Navon and Ciechanover 2009).…”